PERIODIC-ACID SCHIFF (PAS) REACTION

PERIODIC-ACID SCHIFF (PAS) REACTION

This reaction depends on the liberation of carbohydrate radicals from compounds, such as glycogen; and their oxidisation to aldehydes by the Schiff reagent. Polysaccharides, mucopolysaccharides, glycoproteins and mucoproteins are PAS positive. 

A positive reaction is indicated by a bright red colour. In blood cells a positive reaction usually denotes the presence of glycogen. Developing granulocytes react positively at all stages of development.

Mature polymorphonuclear neutrophils react most strongly showing large amounts of magenta coloured granules in their cytoplasm. Myelocytes contain fewer positively staining granules with a pale pink cytoplasm. In eosinophils the background cytoplasm is positive but the large specific granules show a negative reaction.

contain a small amount of fine, scattered positively staining material. The cytoplasm of normoblast does not normally stain at any stage of development. Platelets are positive and megakaryocytes also show a strong positivity. The positive granules are enormous in lymphoblasts in acute lymphocytic leukaemia.

The erythroblasts react positively in diseases like erythroleukaemia, thalassaemia, iron deficiency anaemia, sideroblastic anaemia, myelosclerosis. 

Specimen Thin film prepared with peripheral blood or bone marrow. 

Reagents

1. Periodic acid 10 g per litre. 

2. Schiff''s reagent 

-Dissolve 1 gm basic fuchsin in boiling water and dilute upto 400 ml. Cool the solution to 50°C and filter. To the filtrate add 1 ml of thionyl chloride and allow to stand in the dark for 12 hours.

-Add 2 g of activated charcoal, shake for 1 minute and filter. Store the solution in the dark at 0 to 4°C. Filter before use. 

3. Aqueous haematoxylin: Dissolve 2 g

haematoxylin in 100 ml distilled water, 

Technique

(i) Fix the blood film in methanol for 5 to 15 minutes.

(ii) Wash in running tap water for 15 minutes.

(iii) Allow it to stand in periodic acid solution for 10 minutes and immerse in Schiff's reagent in a coplin jar for 30 minutes.

(iv) Rinse the slide in tap water and then in distilled water for five minutes. Counterstain with aqueous haematoxylin for 10-15 minutes.

(v) Dry the film in the air, mount in a neutral mountant such as DPX, and examine.